Diabetes Volume 67, April 2018 581 Long Noncoding RNA lncSHGL Recruits hnRNPA1 to Suppress Hepatic Gluconeogenesis and Lipogenesis Junpei Wang,1,2 Weili Yang,1,2 Zhenzhen Chen,1 Ji Chen,1 Yuhong Meng,1 Biaoqi Feng,1 Libo Sun,3 Lin Dou,4 Jian Li,4 Qinghua Cui,2 and Jichun Yang1 Diabetes 2018;67:581–593 | https://doi.org/10.2337/db17-0799 Mammalian genomes encode a huge number of long a potential strategy for the treatment of type 2 diabetes noncoding RNAs (lncRNAs) with unknown functions. and steatosis. This study determined the role and mechanism of a new lncRNA, lncRNA suppressor of hepatic gluconeogenesis and lipogenesis (lncSHGL), in regulating hepatic glucose/ The human and other mammalian genomes produce a huge lipid metabolism. In the livers of obese mice and patie- number of transcripts (1,2), 80–90% of which are not tra- nts with nonalcoholic fatty liver disease, the expression ditional protein-coding RNAs and termed long noncoding levels of mouse lncSHGL and its human homologous RNAs (lncRNAs) with the length .200 nucleotides (3–6). lncRNA B4GALT1-AS1 were reduced. Hepatic lncSHGL To date, a great number of lncRNAs have been identified in METABOLISM restoration improved hyperglycemia, insulin resistance, the tissues and circulation of humans and other mammals and steatosis in obese diabetic mice, whereas hepatic (6–10), and dysregulated lncRNA expression profiles are lncSHGL inhibition promoted fasting hyperglycemia and involved in the pathogenesis of many diseases (6,11–15). lipid deposition in normal mice. lncSHGL overexpression So far, the lncRNAs with function annotations are very few increased Akt phosphorylation and repressed gluconeo- in number. Clearly, lncRNA is a huge treasure vault full of genic and lipogenic gene expression in obese mouse unknown but exciting molecules awaiting exploration. livers, whereas lncSHGL inhibition exerted the opposite There has been increasing evidence that lncRNAs regu- effects in normal mouse livers. Mechanistically, lncSHGL late glucose and lipid metabolism. Dysregulated lncRNA recruited heterogeneous nuclear ribonucleoprotein A1 expression profile is associated with islet dysfunction in (hnRNPA1) to enhance the translation efficiency of CALM humans (16). Knockdown of lncRNA TUG1 causes pancre- mRNAs to increase calmodulin (CaM) protein level without b affecting their transcription, leading to the activation of atic -cell dysfunction (17). lncRNA H19 regulates glucose the phosphatidyl inositol 3-kinase (PI3K)/Akt pathway and metabolism by functioning as a sponge for microRNA let-7 fi repression of the mTOR/SREBP-1C pathway indepen- (18,19). lncRNAs long noncoding liver-speci ctriglyceride dent of insulin and calcium in hepatocytes. Hepatic regulator (lncLSTR) and MEG3 also regulate hepatic glu- hnRNPA1 overexpression also activated the CaM/Akt cose and lipid metabolism (20,21). On one hand, the effects pathway and repressed the mTOR/SREBP-1C pathway and mechanisms of the reported lncRNAs in glucose and to ameliorate hyperglycemia and steatosis in obese mice. lipid metabolism in various tissues need further valida- In conclusion, lncSHGL is a novel insulin-independent tion (16,18–21). On the other hand, to further characterize suppressor of hepatic gluconeogenesis and lipogene- new lncRNAs that regulate glucose and lipid metabolism sis. Activating the lncSHGL/hnRNPA1 axis represents is also of great importance. Particularly, identifying new 1Department of Physiology and Pathophysiology, School of Basic Medical Scien- Corresponding author: Jichun Yang, [email protected], or Qinghua Cui, ces, Key Laboratory of Molecular Cardiovascular Sciences of the Ministry of [email protected]. Education, Center for Non-coding RNA Medicine, Peking University Health Sci- Received 9 July 2017 and accepted 16 January 2018. ence Center, Beijing, China This article contains Supplementary Data online at http://diabetes 2Department of Biomedical Informatics, School of Basic Medical Sciences, Key .diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0799/-/DC1. Laboratory of Molecular Cardiovascular Sciences of the Ministry of Education, Center for Non-coding RNA Medicine, Peking University Health Science Center, J.W. and W.Y. contributed equally to this work. Beijing, China © 2018 by the American Diabetes Association. Readers may use this article as 3Beijing You An Hospital, Capital Medical University, Beijing, China long as the work is properly cited, the use is educational and not for profit, and the 4Key Laboratory of Geriatrics, Beijing Institute of Geriatrics & Beijing Hospital, work is not altered. More information is available at http://www.diabetesjournals Ministry of Health, Beijing, China .org/content/license. 582 lncSHGL Affects Gluconeogenesis and Lipogenesis Diabetes Volume 67, April 2018 lncRNAs that regulate hepatic gluconeogenesis will shed viral injection using different sets of mice. On the 9th day, light on the pathogenesis of type 2 diabetes. themiceweresacrificed on fed state. The serum and tissues A great number of new lncRNAs were identified in mouse were collected for biochemical analyses. liver and plasma in our previous studies (7,8). Eleven lncRNAs dysregulated in liver after ischemia/reperfusion Knockdown of lncSHGL in C57BL/6 Mouse Livers injury (IRI) had been validated and identified with high Stealth small interfering (si)-lncSHGL were synthesized by expression (7,8). Among these 11 lncRNAs, AK143693 is Invitrogen (sequences provided in Supplementary Table 1). a nonsecretory lncRNA that exhibits high expression in The siRNA mixture was injected into C57BL/6 mice via m mouse liver with unknown function(s) (7,8). We found in tail vein (2.5 mg/kg body weight in 100 L sterile saline) the preliminary experiment that AK143693 silencing in- (23,24), the same dose of scrambled siRNA (Invitrogen) was creased lipid deposition in liver after IRI (Supplementary used as the control. OGTTs were performed 72 h after the fi Fig. 1A and B), suggesting it may play roles in regulating siRNA injection. On the 4th day, the mice were sacri ced hepatic glucose/lipid metabolism. The current study revealed for experimental analyses. that lncRNA AK143693 functions as a novel suppressor Cell Culture of hepatic gluconeogenesis and lipogenesis (SHGL) and is Cell lines or primary mouse hepatocytes were infected with renamed as lncSHGL. 50 multiplicity of infection of Ad-lncSHGL or Ad-GFP for lncSHGL expression was reduced in obese mouse livers. 24 h. For insulin-stimulation experiments, infected cells lncRNA B4GALT1-AS1, the human homologous sequence of were serum starved for 12 h, followed by treating with mouse lncSHGL, was also reduced in human livers with 100 nmol/L insulin for 5 min. For inhibition of phospha- steatosis. Hepatic lncSHGL overexpression suppressed glu- tidyl inositol 3-kinase (PI3K) or P2 receptors or calcium coneogenesis and attenuated hyperglycemia and fatty liver in signaling, infected cells were treated with 1 mmol/L mice fed a high-fat diet (HFD), whereas hepatic lncSHGL wortmannin, 50 mmol/L pyridoxalphosphate-6-azophenyl- repression promoted hyperglycemia and lipid deposition in 2’,4’-disulfonic acid, 50 mmol/L suramin, 100 mmol/L chlor- normal mice. Mechanistically, lncSHGL recruited heteroge- promazine (CPZ), an inhibitor of CaM (25), 10 mmol/L neous nuclear ribonucleoprotein A1 (hnRNPA1) to enhance nifedipine, or 10 mmol/L 2-aminoethoxydiphenyl borate calmodulin (CaM) mRNAs translation. An increase in the for 1 h before experimental assays. For depleting extracel- fi CaM protein level nally suppressed gluconeogenic and lular Ca2+, infected cells were treated with Ca2+-free me- lipogenic pathways in an insulin- and calcium-independent dium plus 0.5 mmol/L EGTA for 2 h, and chlorpromazine manner in hepatocytes. was added 1 h before experimental assays. Plasmid Overexpression of Genes in Hepatocytes RESEARCH DESIGN AND METHODS Cells plated in six-well plates were transfected with 5 mg Experimental Animals plasmid for 24 h. Plasmids expressing human CALM1-2 and Male C57BL/6 mice (8 to 10 weeks old) were fed a 45% HFD hnRNPA1 plasmids were purchased from OriGene (CALM1, for 3 months to induce diabetic and steatotic phenotypes Cat No. SC115829) and Vigene Biosciences China (CALM2, Cat db db (22,23). The study also used 10- to 16-week-old male / No, CH809926; hnRNPA1, Cat No CH877838), respectively. mice on a BKS background. All animal experimental proto- cols complied with the Animal Management Rules of the Determination of Gene Expression at mRNA and Protein Ministry of Health of the People’s Republic of China and Levels the Peking University Guide for the Care and Use of the Target gene mRNA level was normalized to that of b-actin Laboratory Animals. in real-time PCR assays. Each sample was assayed in dupli- cate in each experiment. All primer sequences are provided Antibodies in Supplementary Table 2. The protein levels were analyzed Anti-phosphorylated (p)Akt (phosphorylation at Ser473 by immunoblotting assay. For protein blot quantitation, each site) and Akt antibodies were purchased from CST. Other protein (nonphosphorylated) was first normalized to the antibodies were obtained from Santa Cruz Biotechnology, corresponding housekeeping protein b-actin, and then CST, or other commercial companies. the value was normalized to the control value in each experiment. For quantitation of phosphorylated protein, Adenoviral Overexpression of lncSHGL in HFD Mouse phosphorylated protein was first normalized to the corre- Livers sponding total protein and then was normalized to the